Cultivation of micro-organisms on hydrocarbons

ABSTRACT

Protein concentrates are obtained in a process which includes: 1. Cultivating a hydrocarbon-consuming micro-organism in the presence of an aqueous nutrient medium, a hydrocarbon and a gas containing free oxygen; 2. CONTACTING THE CULTIVATED MICRO-ORGANISM FROM &#39;&#39;&#39;&#39;1&#39;&#39;&#39;&#39; AT A TEMPERATURE BELOW THAT WHICH SUPPORTS ACTIVE FERMENTATION OF THE MICRO-ORGANISM WITH A GAS CONTAINING FREE OXYGEN IN THE PRESENCE OF AN AQUEOUS NUTRIENT MEDIUM CONTAINING DIPHENYL DISULFIDE AND A GAS CONTAINING FREE OXYGEN IN THE ABSENCE OF ADDED HYDROCARBON; AND 3. RECOVERING FROM &#39;&#39;&#39;&#39;2&#39;&#39;&#39;&#39; A MICRO-ORGANISM HAVING A SULFURCONTAINING AMINO ACID, E.G., CYSTINE AND METHIONINE, CONTENT GREATER THAN THAT OF THE MICRO-ORGANISM OBTAINED IN &#39;&#39;&#39;&#39;1.

United States Patent 1 1 Bunting et a1.

1541 CULTIVATION 0F MICRO- ORGANISMS 0N HYDROCARBONS ["i'5j""T1Vefif6rsi Pamela M. Bunting, Cheswick, Pa.; 1 William W. Leathen,Wexford, Pa.

[73] Assignee: Gulf Research & Development Co.

22 Filed: June2l,l97l 21 Appl/No; 155,275

511 Int. Cl ..C12b 1/00, Cl2c 11/00 3,620,927 ll/l97l Leathen ..195/82 11 Jan. 30, 1973 Primary Examiner-Alvin E. Tanenholtz AssistantExaminer-YR. B. Penland Attorney-Meyer Neishloss et al.

571 ABSTRACT Protein concentrates are obtained in a process whichincludes:

1, Cultivating a hydrocarbon-consuming micro-organism in the presence ofan aqueous nutrient medium, a hydrocarbon and a gas containing freeoxygen;

2. contacting the cultivated micro-organism from 1 at a temperaturebelow that which supports active fermentation of the micro-organism witha' gas containing free oxygen in the presence of an aqueous nutrientmedium containing diphenyl disulfide and a gas containing free oxygen inthe absence of added hydrocarbon; and

3. recovering from 2 a micro-organism having a sulfur-containing aminoacid, e.g., cystinc and methionine content greater than that of themicro-organism obtained in l."

19Claims, No Drawings CULTIVATION F MICRO-ORGANISMS ON HYDROCARBONS Thisinvention relates to a process for cultivating micro-organisms onhydrocarbons and more particularlyto a process wherein propagation ofthe micro-organisms is conducted under conditions to increase theproportion of sulfur-containing amino acids in the product.

Critical food shortages for both animals and humans in some parts of theworld is a problem of growing concern. The use of fertilizers andimproved farming techniques have greatly increased crop return per acreof cultivated ground. While increased crop yields has resulted in morefood for more people throughout the world, there are still an alarmingnumber of people who are suffering from malnutrition. To alleviatemalnutrition, protein and vitamin food supplements have been developedfor both animal and human consumption. Protein concentrates for foodsupplements that are commercially available include fish meal, peanutmeal, cottonseed meal, soybean meal and micro-organisms such asbacteria, m'olds, yeasts and the like.

Micro-organisms, because of their high rate of multiplication infermentation processes, have received much attention as a source of highquality protein. The ability ofmicro-organisms to metabolizecarbohydrates is well-known. it is equally well-known, however, thatcarbohydrates are relatively expensive raw materials if the desired endproduct is a relatively inexpensive micro-organism. Because of theabundance of relatively inexpensive crude oil deposits, a considerable.

amount of experimental work has been conducted utilizing hydrocarbonsderived from petroleum as the sole source of carbon for the growth ofmicro-organisms. A number of micro-organisms and particularly yeasts andbacteria have been found to grow on substrates containing petroleumhydrocarbons varying from normally gaseous hydrocarbons through normallyliquid hydrocarbons and hydrocarbons that are solid under normalatmospheric conditions. When a hydrocarbon-consuming micro-organism isgrown in a substrate containing a hydrocarbon under favorablegrowth-inducing conditions, the micro-organism product has a chemicalcomposition essentially the same as the micro-organism used as seed inthe fermentation process. v

While it is generally desirable to employ fermentation conditions whichprevent the growth of a micro-organism which is a variant from the seedmicro-organism it isfrequently desirable to obtain a product which ismore nutritious than the micro-organism used as seed. Thus, for example,it is desirable in some instances to alter the amino acid profile of theproduct and to increase the proportion of the sulfur-containing aminoacids, i.e., cystine and methionine, in the product.

In accordance with the present, invention, a process is provided forpropagating a micro-organism on a hydrocarbon-containing substratewherein the'proportion of sulfur-containing amino acids in themicro-organism product is increased. In brief, the process comprisescultivating a hydrocarbon-consuming micro-organism in 'a fermentationstage and thereafter subjecting the micro-organism product obtained inthe fermentation stage to a maturation stage in the presence of anaqueous nutrient medium to which a small amount of diphenyl disulfidehas been added. In the fermentation stage, the hydrocarbon-consumingmicroorganism is cultivated at an active fermentation temperature in thepresence of an aqueous nutrient medium, a hydrocarbon and a gascontaining free oxygen. in the maturation stage, the micro-organismproduct obtained in the fermentation stage is contacted with a gascontaining free oxygen in the presence of an aqueous nutrient mediumwhich contains diphenyl disulfide and in the absence of addedhydrocarbon at a temperature below the temperature employed in theactive fermentation stage of the process. Maturation of themicro-organism can be conducted in the fermentation vessel or, ifdesired, the micro-organism product of the fermentation stage can beseparated from the fermentation mass (brew) and thereafter contacted ina separate vessel with a gas containing free oxygen in the presence ofthe aqueous nutrient medium which contains a small amount of diphenyldisulfide dissolved in the nutrient. The product obtained in thematuration stage of the process has a sulfur-containing amino acidcontent greater than the sulfur-containing amino acid content of theproduct from the fermentation stage. in a preferred embodiment of theinvention the product obtained in the maturation stage also has aprotein content greater than the protein content of either the productfrom the fermentation stage or the product from a maturation stageutilizing no diphenyl disulfide.

We have found that a micro-organism product which has an increasedproportion of sulfur-containing amino acids in its amino acid profilecan be obtained in a process which comprises cultivating, in afermentation stage, a hydrocarbon-consuming micro-organism at an activefermentation temperature of about 25 to about 40 C. in the presence ofan aqueous nutrient medium, a hydrocarbon and a gas containing freeoxygen to obtain a cultivated micro-organism and contacting, in amaturation stage, the cultivated micro-organism obtained in thefermentation stage at a temperature below that which supports activefermentation of the microorganism with a gas containing free oxygen inthe presence of an aqueous nutrient medium containing a small amount ofdiphenyl disulfide dissolved therein and in the absence of addedhydrocarbon whereby there is obtained a micro-organism product having asulfur-containing amino acid content greater than that of the cultivatedmicro-organism obtained in the fermentation stage. The temperature atwhich maturation is conducted in accordance with the process of theinvention depends somewhat upon the particular microorganism beingcultured and upon the nutrient medium employed. If the micro-organism isactively cultivated in the fermentation stage at about 25, maturationshould be effected at a temperature below 25 C. and preferably at atemperature within the range of about 10 to about 20 C. if themicro-organism is actively cultivated in the fermentation stage at about35 C., maturation should be effected at a temperature below 35 C. If themicro-organism is actively cultivated in the fermentation stage at about40 C., the maturation should be effected at a temperature below 40 C. Itis preferred to effect maturation at a temperature which assimilatecarbon from hydrocarbons or those which have been adapted to assimilatecarbon from hydrocarbons including molds, bacteria and yeasts. Typicalexamples of molds are those of the family Aspergillaceae, suitablegenera of which are Penicillium and Aspergillus. Specific examples ofmolds within these genera are Penicillium rocqueforti, Penicilliumglaucum, Penicillium chrysogenum, Penicillium patulum, Penicilliumnotatum, Penicillium espansum, Aspergillus fumigatus, Aspergilluscarbonarious, Aspergillus niger, Aspergillus flavus, Aspergillusterreus, and 'Aspergillus versicolor.

Bacteria which can be employed in the process of the invention are thosewithin the group consisting of Pseudonomadales, Eubacteriales, andActinomycetales. The bacteria which are employed are preferably of thefamilies Bacillaceae and Pseudonomadaceae, preferred species beingBacillus megaterium, Bacillus subtilis, and Pseudomonas aeruginosa.

Yeasts which can be employed in the process of the invention arepreferably those of the family Cryptococcaceae and especially of thesub-family Cryptococcoideae. Other yeasts can also be employed such asthose of the family Endomycetaceae and especially of the sub-familySaccharomycetoideae. Preferred genera of the Cryptococcoideae sub-familyare Torulopsis and Candida. Preferred strains of yeast are Candidautilis, Candida rugosa, Candida lipolytica Candida tropicalis, andTorulopsis colliculosa. Of these yeasts, a strain of Candida tropicalisis preferred, particularly Candida tropicalis, strains CS-8-l 7 andCs-9-5 which are essentially identical strains which have been isolatedfrom petroleum-soaked soils. Candida tropicalis, strains CS-8-l7 andCS-9-5 have been deposited in the American Type Culture Collection inRockville, Maryland. These strains have been assigned the ATCC numbers20021 and 20326, respectively. The advantage of utilizing amicro-organism which has been isolated from an oil-soaked soil is thatthe organism is already adapted to metabolize hydrocarbons so that aninitial hydrocarbon-adaptation procedure is not necessary. If themicro-organism, particularly yeast, has been grown in a carbohydrateenvironment, it is usually necessary to adapt the organism to grow oncarbon supplied by the hydrocarbon. This procedure may require aprolonged period of time. Even yeasts which have been isolated fromoil-soaked soil may require an adaptation procedure to adapt the yeastto grow on the particular hydrocarbon which is intended to be used asfeedstock in the fermentation process.

Inasmuch as micro-organisms are made-up of living cells, their growth,as is true with other living organisms, depends upon an adequate supplyof carbon, hydrogen, oxygen, nitrogen and trace amounts of otherelements including sodium, potassium, magnesium and iron. Carbon isrequired for growth and energy. Nitrogen is required for synthesis ofprotein and other nitrogenous materials. Other elements are required formineral structure of the cell. In the present process, carbon andhydrogen are supplied by the hydrocarbon; oxygen is supplied by theintroduction of air; and nitrogen is supplied through the use ofammonium or other nitrogenous inorganic salts in the aqueous nutrientmedium. Trace quantities of other elements necessary for growth of themicro-organism may be supplied as impurities in the inorganic salts orthese elements may be added directly in extremely small amounts.Frequently, sufficient quantities of the trace elements are present intap water.

The hydrocarbon which is utilized as the source of carbon and hydrogenfor the micro-organism is a saturated or unsaturated aliphatichydrocarbon having up to 30 or more carbon atoms per molecule. Apreferred hydrocarbon feedstock is a petroleum fraction, especially apetroleum fraction consisting essentially of a mixture of straight chainhydrocarbons. The straight chain hydrocarbons can be present as olefins,paraffins or a mixture containing both olefins and paraffins. Examplesof individual hydrocarbons which can be used are n-pentane, l-pentene,n-hexane, l-hexene, n-heptane, l-heptene, n-octane, n-decane, l-decene,ndodecane, l-dodecene, n-tetradecane, l-tetradecene, n-hexadecane,n-octadecane, n-eicosane, ntetracosane, n-triacontane and the like.Those hydrocarbons which are liquid at the fermentation conditionsemployed are preferred. While the individual hydrocarbons can be used,we prefer, for economic reasons to use mixtures of hydrocarbons. Thus,we may use kerosene, gas oil, middle distillate fractions, slack wax andthe like. Good results have been obtained with hydrocarbon mixturescomprising naphtha (C -C nparaffins (C -C n-paraffins (C -C n-paraffins(C -C n-paraffins (C C alpha olefins (C -C kerosene (C -C and slack wax(C -C alone and in admixture with naphtha. The amount of hydrocarbonemployed is that amount required to provide suffrcient carbon to supportgrowth of the micro-organism during the fermentation period. All thehydrocarbon can be added at the beginning of the fermentation period,but in a continuous process the hydrocarbon is preferably addedcontinuously or incrementally as the fermentation proceeds. It ispreferred to employ only that amount of hydrocarbon required to effectthe desired growth in order to avoid subsequent separation difficulties.In a continuous process, pure n-paraffins are added at a rate of about0.2 to about 5 grams of paraffin per liter of the fermentation mass(brew) per hour. When a hydrocarbon mixture is employed, the mixture isadded at a rate proportioned to the normal paraffin content, so that theamount of normal paraffin added is about 0.2 to about 5 grams ofparaffin per liter of brew in the fermentor per hour. In a batchprocess, the hydrocarbon comprises about 1 to about 10 percent by weightof the fermentation mass (brew), preferred amounts for most favorablegrowth of Candida tropicalis (CS-8-l7 and CS-9-5) being about 2 to about5 percent by weight.

Oxygen, as disclosed hereinabove, is one of the essential elementsrequired to promote the growth of the micro-organism. While pure oxygencan be employed, we prefer for economic reasons to supply the oxygen asair. In order to effect an optimum growth of the microorganism, the airshould be finely dispersed through the substrate preferably withagitation at a rate sufficient to form a vortex in the liquid. Dependingupon the design of the fermentor, various air introducing means can beused including single orifice, half and full ring types with openingsfor air discharge directed upwardly and/or downwardly, and sinteredglass percolator types with various impellers for lifting air.Impellers, when employed, can be rotated at rates from 40 to 1,000 rpmor more, the particular rate being chosen to create a vortex in theliquid. Regardless of the type of air introduction means employed,operation should be such as to avoid excessive foaming since foamingtends to entrap the micro-organism and remove it from the source ofsoluble nutrients required in its growth. While the amount of airemployed depends somewhat on the size and design of the fermentor, goodresults are obtained in 14-liter fermentors containing 7 liters of brewwhen employing sterile air at rates of 2 to liters per minute.

The make-up of the aqueous nutrient medium which we employ in thefermentation stage of the process will vary to some extent dependingupon the type of microorganism used and the type of hydrocarbon. Ingeneral, the aqueous nutrient medium in the fermentation stage comprisesa mixture of mineral salts which furnish ions of ammonium, nitrate ornitrite, potassium, ferrous or ferric, calcium, magnesium, phosphate,sulfate as well as ions of trace elements including zinc, manganese,copper and molybdenum. Inasmuch as water is included in the nutrientmixture, many of the mineral salts can be incorporated into thesubstrate in sufficient quantity through the use of tap water. It isdesirable,

however, to add the salts to the mixture to insure their presence insufficient quantity for growth of the microorganism. The nutrientmixture consists primarily of water, which may constitute about 50 to 99percent by weight or more of the total nutrient mixture. Generally, thewater is employed in an amount normally used in microbial synthesis. Ingeneral, in a continuous process, the aqueous nutrient medium is addedat a rate of about 0.10 to 0.30 liter per liter of brew in the fermentorper hour, a preferred rate for most favorable growth of Candidatropicalis (CS-8-l7 and CS-95) being about 0.15 to about 0.25 liter perliter of brew per hour.

A typical mineral salts medium referred to as Media Formulation A forthe growth of yeasts of the genus Candida, for example Candidatropicalis, in the process of the invention has the followingcomposition:

MEDIA FORMULATION A Macro Ions, (g/l):

n Distilled water, sufficient to form l liter of solution If desired,the above Media Formulation A can be enriched with vitamins other thanBiotin in which case the amount of Biotin can be reduced. Thus, suitablevitamin enriched aqueous nutrient mediums for use in the process of theinvention may have the following composition:

MEDIA FORMULATION A (Vitamin enriched) Single Double Components StrengthStrength Macro Ions, (g/l):

Ammonium sulfate, (NII ),SO, 0.66 0.66 Magnesium sulfate, MgS0 7H,0 0.51.0 Sodium chloride, NaCl 0.1 0.2 Calcium chloride, CaCl,-2H,O 0.\ 0.2Potassium dihydrogen phosphate, KH PO 1.0 2.0 Trace Elements, (pg/l):

Boric acid, H 80, 500 1000 Copper sulfate, CuSO -5H 0 80 Potassiumiodide, KI I00 200 Ferric chloride, FeCl,-6H,O 200 400 Manganesesulfate, MnS0,'I-l,0 400 800 Sodium molybdate, Na,MoO *H O 200 400 Zincsulfate, ZnSOflI-LO 400 800 Vitamins, (pg/l):

Biotin 2 4 Calcium pantothenate 400 800 Folic acid 2 4 Inositol 2,0004,000 Niacin 400 800 p-Aminobenzoic acid 200 400 Pyridoxin hydrochloride400 800 Riboflavin 200 400 Thiamin hydrochloride 400 400 Distilledwater, sufficient to form 1 liter of solution Another typical mineralsalts medium referred to as Media Formulation B which can be used as anutrient in the process of the invention has the following composition:

Media Formulation B Diammonium phosphate, (NHJ HPO l 3 g. Potassiumdihydrogen phosphate, KH PO, l.3 g. Magnesium sulfate, MgSO '7I-I O 0.2g. Calcium chloride, CaCl,-2H,O 0.02 g. Ferric chloride, FeCl,-6H,Otrace Yeast extract 1.0 g. Distilled water, sufficient to form I literof solution .The aqueous'nutrient medium utilized in the maturationstage of the present process comprises an aqueous nutrient medium asdefined above to which has been added a small amount of diphenyldisulfide for assimilation by the micro-organism. The aqueous nutrientmedium to which the diphenyl disulfide is added can be either freshlyprepared medium similar to that used in the fermentation stage asdescribed hereinabove or aqueous nutrient medium which previously hasbeen used in the fermentation stage. Thus, in a preferred embodiment ofthe invention, the entire contents of the fermentation vessel aretransferred to a maturation vessel wherein the diphenyl disulfide isadded. If desired, a small amount of freshly prepared aqueous nutrientmedium can be added to make up for any loss encountered in the transferor to'make up for nutrients which have been depleted in the fermentationstage.

The diphenyl disulfide can be added to the aqueous nutrient medium byany means which assures the formation of a homogeneous solution. Inorder to facilitate dissolution of diphenyl disulfide in the aqueousnutrient medium, we prefer to prepare a stock solution of diphenyldisulfide, which stock solution is thereafter added to the aqueousnutrient medium. In preparing a stock solution of diphenyl disulfide, wecan use any organic solvent which will dissolve the diphenyl disulfideand which is sufficiently miscible with the aqueous nutrient medium toform a homogeneous solution therewith. Chloroform is an example of onesolvent which can be used in preparing a stock solution of diphenyldisulfide for use in the process of the present invention.

The amount of diphenyl disulfide which effects an increased proportionof the sulfur-containing amino acids in the matured product is a smallamount, and generally is expressed in terms of parts of sulfur permillion parts of aqueous nutrient medium. In general, the diphenyldisulfide is employed in an amount such that the sulfur supplied by saiddiphenyl disulfide does not exceed 50 parts by weight of sulfur permillion parts by weight of aqueous nutrient medium. A preferred range ofsulfur is about 10 to about 35 ppm. An especially preferred amount ofdiphenyl disulfide is that amount required to give 25 ppm of sulfur.

During the course of the growth of a hydrocarbon-assimilatingmicro-organism on a hydrocarbon substrate in the presence of an aqueousmineral nutrient medium and an oxygen containing gas, oxygen is absorbedand carbon dioxide is liberated, and acidic substances, principallyfatty acids are formed. The net effect of these processes is a reductionof the pH of the aqueous nutrient medium. Thus, to prevent a build-up ofacidity which adversely affects the growth of the micro-organism, it isessential to add an alkaline material to restore the pH of the aqueousnutrient medium to a desired level. If the pH is not maintained at adesired level, the growth of the micro-organism ceases, that is cellulardensity no longer increases so that a stationary growth phase isencountered.

The optimum pH of the aqueous nutrient medium depends somewhat upon thenature of the substrate and the particular micro-organism beingcultured. The pH is usually within the range of about 1.5 to about 8.

With mineral salts substrates, the optimum pH for most yeast cultures isa pH of about 5. When employing a yeast nitrogen base substrate, optimumgrowth for a yeast of the strain of Candida tropicalis occurs at a pH ofabout 2 to 5, a pH of about 3 being preferred. While optimum ranges ofpH for molds is also within the range of about 2 to 5, bacteria usuallyrequires a higher pH in the order of about 6 to 8. In order to maintainthe pH at any desired level, we may add to the aqueous nutrient medium,either continuously or in separate increments, any suitable alkalinematerial such as sodium hydroxide, potassium hydroxide, disodiumhydrogen phosphate, ammonium hydroxide and ammonia.

The optimum temperature for the growth of the micro-organism isdependent upon the particular organism employed but with a yeast willusually be within the range of about 25 to about 40 C. When using astrain of Candida trapicalis the preferred temperature range is about25to about 35C.

Micro-organisms grown under controlled conditions in the presence ofvariable amounts of all nutrients required to support growth and underenvironmental conditions favorable to growth typically grow in acharacteristic pattern which may be designated as follows:

1. Initial stationary phase-In this phase, the number of micro-organismsremains constant. a

2. Lag phase-During this period, the rate of multiplication increaseswith time.

3. Logarithmic growth phase-The rate of multiplication remains constant;the generation time is the same throughout the period.

4. Negative growth phase-During this phase, the rate of multiplicationdecreases and the average generation time increases. The organismscontinue to increase in number, but at a slower rate than during thelogarithmic phase.

5. Maximum stationary phase--The number of living organisms remainsconstant, i.e., the death rate equals the rate of reproduction.

6. Accelerated death phase-The number of microorganisms declines withincreasing rapidity. The average rate ofdeath increases to a maximum.

7. Logarithmic death phase-In this period, the rate of death isconstant.

According to the present invention active fermentation of themicro-organism in the fermentation stage is interrupted at the end ofthe logarithmic growth phase and the cultivated micro-organism soobtained is subjected to treatment in a maturation stage at atemperature below that which supports active fermentation in thepresence of the aqueous nutrient medium which contains diphenyldisulfide. During the treatment in the maturation stage, thesulfur-containing amino acid content of the product is increased.

At the conclusion of the maturation stage in the process of theinvention, the product is separated from the diphenyldisulfide-containing nutrient medium and then washed one to three timeswith water and finally dried under conditions sufficiently mild to avoidautolysis but under conditions sufficiently severe to assure recovery ofa non-viable micro-organism containing not more than about 10 percentmoisture, usually less than 1 percent moisture. With bacteria, thedrying temperature may be in the order of about C. The dryingtemperature for most yeasts to ensure the recovery of non-viable cellsin an oven is within the range of about 50 to about C. If spray dryingis employed, the temperature of the dryer may be in the order of aboutC. without adversely affecting the quality of the yeast. In drying astrain of Candida tropicalis, we have recovered non-viable cells byemploying a drying temperature of 60 C. in an oven. 7

In order to illustrate the improved results obtained in accordance withthe process of the invention, comparative examples are set forthhereinafter wherein propagation of the micro-organism is effected incombined fermentation-maturation stages with and without the addition ofdiphenyl disulfide in the maturation stage. In the comparative examples,we have utilized a hydrocarbon-consuming yeast as the micro-organism,i.e., Candida tropicalis, strain CS-9-5 (ATCC 20326). This yeast wasisolated through an enrichment culture procedure from oil-soaked soiladjacent to operating oil wells located in Pennsylvania. Themicro-organism was characterized and identified in accordance with theclassification in The Yeasts by J. Lodder and N. J. W. Kreger-Van Rij,North Holland Publishing Co., Amsterdam, 1952, lnterscience Publishers,lnc. New

York. The micro-organism was found to be identical in n-Paraffin WeightPercent C trace C 6.8 C, 35.2 0., 32.1 C 25.8

EXAMPLE I This example illustrates the growth of a hydrocarhon-consumingmicro-organism in a fermentation stage followed by maturation in aseparate vessel with and without (for control purposes) the addition ofdiphenyl disulfide.

Seven liters of aqueous nutrient medium referred to hereinabove as MediaFormulation A are introduced into a 14-liter Pyrex glass fermentor jarequipped with a stainless steel head assembly. The head assemblycontains ports for the addition of nutrients and removal of samples, anagitator shaft, an air sparger line, baffles and a thermometer well. Thefermentor jar is placed in a water bath which is adjusted to maintainthe fermentation medium at 28ilC. The fermentor is equipped withimpellers connected to a drive mechanism capable of rotating theimpellers at rates up to 1,000 rpm. The

the first hour to 6.7 ml in thelast hour. Buring this same 8-hour perioda total of 20.6 ml of an aqueous solution of ammonium hydroxide andammonium sulfate containing about mg of nitrogen per ml are added athourly intervals in increasing increments starting with 1.3 ml in thefirst hour and ending with 4.5 ml in the last hour. The pH of thefermentation mass is maintained at about 3 during the activefermentation period by the addition of ammonium hydroxide. At the end ofthe 8-hour active fermentation period a sample of the fermentation mass(brew) is removed wherein the yeast cells are harvested by filtering,washing with water, refiltering and drying prior to evaluation as toprotein content, amino acid content and amino acid profile.

Separate 600 ml portions of the fermentation mass (brew) removed fromthe fermentor jar are placed in four separate 2-liter, baffled shakeflasks. For control purposes, nothing except the fermentation mass isadded to one of the shake flasks. To the other three shake flasks, thefermentation mass is admixed with separate portions of diphenyldisulfide. To facilitate dissolution of the diphenyl disulfide in thefermentation mass, a stock solution of diphenyl disulfide is prepared bydissolving 6.82 grams of diphenyl disulfide in 100 milliliters ofchloroform. The stock solution thus prepared contains 20 milligrams ofsulfur as diphenyl disulfide per milliliter of stock solution. To thethree shake flasks each of which contains 600 ml of brew are separatelyadded 0.3 ml, 0.75 ml and 1.50 ml of stock solution, or 10 ppm, 25 ppmand 50 ppm sulfur, respectively, as diphenyl disulfide. All of theflasks are then subjected to maturation conditions. These conditionscomprise rotary agitation of the shake flasks at l2.5i2 C. for about 18hours. At the end of the 18-hour maturation period the yeast cells areharvested as above and examined for protein content, amino acid contentand amino acid profile. The results obtained with respect to proteincontent, amino acid content and the amino acid profile for lysine,methionine and cystine are summarized in Table I.

TABLE I After maturation Control, no 10 p.p.m. S 25 p.p.m. S 50 p.p.m SAnalyses, percent by weight of dried After diphenyl as diphenyl asdiphenyl as diphenyl yeast product fermentation disulfide disulfidedisulfide disulfide Crude protein 52. 97 52. 52. 53. 56 48. 88 Lysine 423. 75 3. 99 3. 73 3. 38 Methionine 0. 69 0. 72 0. 74 0. 74 0. 65 Cystine0. 50 0. 58 0. 63 0. 66 0. 59 Combined cystine and methionine 1. 19 1.30 1. 37 1.40 1. 24

This value is considered to be unusually high and is believed to be anexperimental error. The crude protein content after fermentation isusually in the range of about 40 to 50 percent by weight of the driedyeast product.

Active fermentation is conducted for 8 hours at 28:1C. with an aerationrate of about 5 liters per minute at impeller speeds of 500 to 700 rpm.During this 8-hour period a total of 30.6 ml of the abovedescribed C -Cnormal paraffln mixture are added at hourly intervals in incrementsincreasing from 1.9 ml in it will be noted from the data in Table 1 thatthe presence of 10 to 25 ppm of sulfur as diphenyl disulfide in theaqueous nutrient medium during the maturation stage gives a higherproportion of methionine and cystine in the amino acids obtained andthat with 25 ppm of sulfur as diphenyl disulfide there is also anincrease in the crude protein content of the yeast product. While 50ppmof sulfur as diphenyl disulfide gives a combined cystine and methioninecontent which is greater than that of the yeast obtained in thefermentation stage, the combined cystine and methionine content is notas great as the combined cystine and methionine content obtained in thematuration stage with no diphenyl disulfide. In the light of this data,it is evident that the most advantageous results are obtained when usingless than 50 ppm of sulfur as diphenyl disulfide, the optimum resultbeing obtained with about 25 ppm of sulfur as diphenyl disulfide.

EXAMPLE 11 TABLE 11 After Maturation Analyses, Percent by After Control35 ppm S weight of dried yeast Fermen No Diphenyl as Diphenyl producttation disulfide disulfidc Crude Protein 48.56 51.13 47.88 Lysine 3.223.88 4.49 Methionine 0.42 0.49 0.68 Cystine 0.29 0.45 0.59 Combinedcystine and methionine 0.71 0.94 1.27

It will be noted from the data in Table 11 that the presence of 35 ppmof sulfur as diphenyl disulfide in the aqueous nutrient medium duringthe maturation stage gives a higher proportion of lysine, methionine andcystine in the amino acids obtained but that there is a decrease in thecrude protein content of the yeast.

The make-up of Candida tropicalis (CS-9-5) obtained in the process ofthe present invention may vary slightly from one fermentation toanother. A typical chemical composition of the dried product obtainedwhen using 25 ppm of sulfur as diphenyl disulfide during maturation isas follows:

Candida tropicalis (CS-9-5) Percent by weight Total nitrogen 8.57 Crudeprotein 53.56 Total amino acids 41.2

A summary of the amino acid profile for a typical Candida tropicalis(CS-9-5) product of the invention is as follows:

While our invention has been described with reference to variousspecific examples and embodiments, it will be understood that theinvention is not limited to such examples and embodiments and may bevariously practiced within the scope of the claims hereinafter made.

We claim:

1. A process which comprises-cultivating in a fermentation stage ahydrocarbon-consuming micro-organism at an active fermentationtemperature in the presence of an aqueous nutrient medium, a hydrocarbonand a gas containing free oxygen; contacting in a maturation stage thecultivated micro-organism obtained in the fermentation stage at atemperature below that which supports active fermentation of themicroorganism with a gas containing free oxygen in the presence of anaqueous nutrient medium containing a small amount of diphenyl disulfideand in the absence of added hydrocarbon; and recovering a micro-organismproduct having a sulfur-containing amino acid content greater than thatof the cultivated micro-organism obtained in the fermentation stage.

2. A process according to claim 1 wherein said micro-organism is abacterium.

3. A process according to claim 1 wherein said micro-organism is ayeast.

4. A process according to claim 3 wherein the diphenyl disulfide isemployed in an amount sufficient to give about 10 to about 35 parts byweight of sulfur per million parts by weight of aqueous nutrient medium.

5. A process which comprises cultivating in a fermentation stage ahydrocarbon-consuming yeast at an active fermentation temperature ofabout 25 to about 40 C. in the presence of an aqueous nutrient medium, ahydrocarbon and a gas containing free oxygen; contacting in a maturationstage the cultivated yeast obtained in the fermentation stage at atemperature which is 5 to 20 C. below the active fermentationtemperature with a gas containing free oxygen in the presence of anaqueous nutrient medium containing a small amount of diphenyl disulfideand in the absence of added hydrocarbon; and recovering a yeast producthaving a sulfur-containing amino acid content greater than that of thecultivated yeast obtained in the fermentation stage.

6. A process according to claim 5 wherein the diphenyl disulfide isemployed in an amount sufficient to give about 10 to about 35 parts byweight of sulfur per million parts by weight of aqueous nutrient medium.

7. A process according to claim 6 wherein the yeast is of the familyCryptococcaceae.

8. A process according to claim 7 wherein the yeast is of the subfamilyCryptococcoideae.

9. A process according to claim 8 wherein the yeast is of the genusCandida.

10. A process according to claim 9 wherein the yeast is of the strainCandida tropicalis.

11. A process according to claim 10 wherein the yeast is Candidatropicalis, strain CS-9-5 (ATCC 20326).

12. A process according to claim 6 wherein the hydrocarbon is a liquidpetroleum fraction consisting essentially ofa mixture of straight chainhydrocarbons.

13. A process according to claim 12 wherein the liquid petroleumfraction is a mixture of C to C normal paraffins.

14. A process according to claim 6 wherein the pH of the aqueousnutrient medium is within the range of about 1.5 to about 8.

15. A process according to claim 6 wherein the oxygen-containing gas isair.

16. A process which comprises cultivating in a fer mentation stage ahydrocarbon-consuming yeast at an active fermentation temperature ofabout 25 to about 40 C. in the presence of an aqueous nutrient medium, ahydrocarbon and a gas containing free oxygen; contacting in a maturationstage the cultivated yeast obtained in the fermentation stage at atemperature of about 10 to about C. with a gas containing free oxygen inthe presence of an aqueous nutrient medium containing a small amount ofdiphenyl disulfide and in the absence of added hydrocarbon; andrecovering a yeast product having a sulfur-containing amino acid contentgreater than that of the cultivated yeast obtained in the fermentationstage.

17. A process which comprises cultivating in a fermentation stage ahydrocarbon-consuming yeast of the strain Candida tropicalis at atemperature of about to about 35 C. in the presence of an aqueousnutrient medium whose pH is maintained within the range of about 2 toabout 5, a hydrocarbon feedstock comprising a mixture of C to C normalparaffins and air; contacting in a maturation stage the cultivated yeastobtained in the fermentation stage at a temperature of about 10 to about20 C. with air in the presence of an aqueous nutrient medium containinga small amount of diphenyl disulfide and in the absence of addedhydrocarbon feedstock; and recovering a Candida tropicalis producthaving a sulfur-containing amino acid content greater than that of thecultivated yeast obtained in the fermentation stage.

18. A process which comprises cultivating in a fermentation stage ahydrocarbon consuming yeast of the strain Candida tropicalis at atemperature of about 25 to about 35 C. in the presence of an aqueousnutrient medium whose pH is maintained at about 3, a hydrocarbonfeedstock comprising a mixture of C to C normal paraffins and air;contacting in a maturation stage the cultivated yeast obtained in thefermentation stage at a temperature of about 10 to about 20 C. with airin the presence of an aqueous nutrient medium containing about 10 toabout 35 parts by weight of sulfur as diphenyl disulfide per millionparts by weight of aqueous nutrient medium and in the absence of addedhydrocarbon feedstock; and recovering a Candida tropicalis producthaving a sulfur-containing amino acid content greater than that of thecultivated yeast obtained in the fermentation stage.

19. A process which comprises cultivating in a fermentation stage ahydrocarbon-consuming yeast of the strain Candida tropicalis at atemperature of about 25 to about 35 C. in the presence of an aqueousnutrient medium whose pH is maintained at about 3, a hydrocarbonfeedstock comprising a mixture of C to C normal paraffins and air;contacting in a maturation stage the cultivated yeast obtained in thefermentation stage at a temperature of about 10 to about 20 C. with airin the presence of an aqueous nutrient medium contaming about 25 partsby weight of sulfur as diphenyl

1. A process which comprises cultivating in a fermentation stage ahydrocarbon-consuming micro-organism at an active fermentationtemperature in the presence of an aqueous nutrient medium, a hydrocarbonand a gas containing free oxygen; contacting in a maturation stage thecultivated micro-organism obtained in the fermentation stage at atemperature below that which supports active fermentation of themicro-organism with a gas containing free oxygen in the presence of anaqueous nutrient medium containing a small amount of diphenyl disulfideand in the absence of added hydrocarbon; and recovering a micro-organismproduct having a sulfur-containing amino acid content greater than thatof the cultivated micro-organism obtained in the fermentation stage. 1.CULTIVATING A HYDROCARBON-CONSUMING MICRO-ORGANISM IN THE PRESENCE OF ANAQUEOUS NUTRIENT MEDIUM, A HYDROCARBON AND A GAS CONTAINING FREE OXYGEN;2. A process according to claim 1 wherein said micro-organism is abacterium.
 2. CONTACTING THE CULTIVATED MICRO-ORGANISM FROM "1" AT ATEMPERATURE BELOW THAT WHICH SUPPORTS ACTIVE FERMENTATION OF THEMICRO-ORGANISM WITH A GAS CONTAINING FREE OXYGEN IN THE PRESENCE OF ANAQUEOUS NUTRIENT MEDIUM CONTAINING DIPHENYL DISULFIDE AND A GASCONTAINING FREE OXYGEN IN THE ABSENCE OF ADDED HYDROCARBON; AND 3.RECOVERING FROM "2" A MICRO-ORGANISM HAVING A SULFURCONTAINING AMINOACID, E.G., CYSTINE AND METHIONINE, CONTENT GREATER THAN THAT OF THEMICRO-ORGANISM OBTAINED IN "1."
 3. A process according to claim 1wherein said micro-organism is a yeast.
 4. A process according to claim3 wherein the diphenyl disulfide is employed in an amount sufficient togive about 10 to about 35 parts by weight of sulfur per million parts byweight of aqueous nutrient medium.
 5. A process which comprisescultivating in a fermentation stage a hydrocarbon-consuming yeast at anactive fermentation temperature of about 25* to about 40* C. in thepresence of an aqueous nutrient medium, a hydrocarbon and a gascontaining free oxygen; contacting in a maturation stage the cultivatedyeast obtained in the fermentation stage at a temperature which is 5* to20* C. below the active fermentation temperature with a gas containingfree oxygen in the presence of an aqueous nutrient medium containing asmall amount of diphenyl disulfide and in the absence of addedhydrocarbon; and recovering a yeast product having a sulfur-containingamino acid content greater than that of the cultivated yeast obtained inthe fermentation stage.
 6. A process according to claim 5 wherein thediphenyl disulfide is employed in an amount sufficient to give about 10to about 35 parts by weight of sulfur per million parts by weight ofaqueous nutrient medium.
 7. A process according to claim 6 wherein theyeast is of the family Cryptococcaceae.
 8. A process according to claim7 wherein the yeast is of the subfamily Cryptococcoideae.
 9. A processaccording to claim 8 wherein the yeast is of the genus Candida.
 10. Aprocess according to claim 9 wherein the yeast is of the strain Candidatropicalis.
 11. A process according to claim 10 wherein the yeast isCandida tropicalis, strain CS-9-5 (ATCC 20326).
 12. A process accordingto claim 6 wherein the hydrocarbon is a liquid petroleum fractionconsisting essentially of a mixture of straight chain hydrocarbons. 13.A process according to claim 12 wherein the liquid petroleum fraction isa mixture of C9 to C13 normal paraffins.
 14. A process according toclaim 6 wherein the pH of the aqueous nutrient medium is within therange of about 1.5 to about
 8. 15. A process according to claim 6wherein the oxygen-containing gas is air.
 16. A process which comprisescultivating in a fermentation stage a hydrocarbon-consuming yeast at anactive fermentation temperature of about 25* to about 40* C. in thepresence of an aqueous nutrient medium, a hydrocarbon and a gascontaining free oxygen; contacting in a maturation stage the cultivatedyeast obtained in the fermentation stage at a temperature of about 10*to about 20* C. with a gas containing free oxygen in the presence of anaqueous nutrient medium containing a small amount of diphenyl disulfideand in the absence of added hydrocarbon; and recovering a yeast producthaving a sulfur-containing amino acid content greater than that of thecultivated yeast obtained in the fermentation stage.
 17. A process whichcomprises cultivating in a fermentation stage a hydrocarbon-consumingyeast of the strain Candida tropicalis at a temperature of about 25* toabout 35* C. in the presence of an aqueous nutrient medium whose pH ismaintained within the range of about 2 to about 5, a hydrocarbonfeedstock comprising a mixture of C9 to C13 normal paraffins and air;contacting in a maturation stage the cultivated yeast obtained in thefermentation stage at a temperature of about 10* to about 20* C. withair in the presence of an aqueous nutrient medium containing a smallamount of diphenyl disulfide and in the absence of added hydrocarbonfeedstock; and recovering a Candida tropicalis product having asulfur-containing amino acid content greater than that of the cultivatedyeast obtained in the fermentation stage.
 18. A process which comprisescultivating in a fermentation stage a hydrocarbon consuming yeast of thestrain Candida tropicalis at a temperature of about 25* to about 35* C.in the presence of an aqueous nutrient medium whose pH is maintained atabout 3, a hydrocarbon feedstock comprising a mixture of C9 to C13normal paraffins and air; contacting in a maturation stage thecultivated yeast obtained in the fermentation stage at a temperature ofabout 10* to about 20* C. with air in the presence of an aqueousnutrient medium containing about 10 to about 35 parts by weight ofsulfur as diphenyl disulfide per million parts by weight of aqueousnutrient medium and in the absence of added hydrocarbon feedstock; andrecovering a Candida tropicalis product having a sulfur-containing aminoacid content greater than that of the cultivated yeast obtained in thefermentation stage.